Protein purification, a process which can be as easy as complex
Protein purification looks simple. For a Histidine or a GST tagged protein as well as for an Immunoglobulin one single affinity step followed by a polishing step is enough to get a pure protein. For all the other proteins it is much more complex. The CEP (Capture – Enhance – Polishing) strategy is a key to solve your purification problem.
For a top level purification some key points have to be kept in mind :
- Protein stability
- What will be the use of the column
- Purification tools
- Proteins characteristics
- Purification planning
Prior any proteins purification we have to keep in mind the proteins could lose their activity entirely or partly. This is due to:
- Proteolytics enzymes
- Structure loss
- Their lost (non-specific adsorption)
- Chemical modifications
To minimize these risks, we have to avoid :
- Multi-step purifications
- Long term storage
- Repeated freeze /thawing steps
and we have to :
- Optimize the purifications conditions
- Work at low temperature
- Optimize the protein concentration
- Eliminate proteases
- Add stabilizers and co-factors
What will the protein be used for?
Following the future use of the protein purifications strategy are different
Following the proteins quantity needed purification tools are different
Chromatography technics that can be used
All the purification technics are usable since all the proteins own negatively charged portions, positively charged portion, hydrophilic, hydrophobic and affinity zones.
1/ Defining the goal
- Trial conditions
- The protein to purify
- The starting material (proteic medium)
3/ Analytical conditions to develop
CEP strategy BioWorks
The CEP strategy: how and which separative technics can be combined
- Combine complimentary technics
- Reduce as much as possible the sample manipulation
3 purification steps must be a maximum. If more, we lose too much target protein. The ratio Purity/cost is no more acceptable.
The purification steps:
“Don´t waste clean thinking on dirty enzymes” Efraim Racker
Keep it simple!
CEP strategy summary
In the Bio-Works CEP strategy the planning will define precisely the purification way of the starting material:
- In which medium is the protein to purify?
- What is the protein (Antibody, recombinant protein, Enzyme…)
- What will the protein be used for?
- Which purifications technics are the more relevant?
The different development steps are used to validate the purification technics, the purification resin and to optimize the protocol (pH, Ionic force, temperature, linear flow rate…)
Access directly to the Bio-Works product range on our website:
- WorkBeads™ 40 Q , WorkBeads™ 40 S, WorkBeads™ 40 DEAE : Ion exchange media (IEX)
- WorkBeads™ 40 SEC, WorkBeads™ 40/100 SEC, WorkBeads™ 40/10 000 SEC : High Performance Size Exclusion Chromatography Media
- WorkBeads™ 40 ACT , WorkBeads™ 40/10 000 ACT: Activated Media for laboratory and process scale Affinity chromatography with User’s choice of ligand
- WorkBeads™ 40 Ni: High throughput agarose media for capture of His-tagged proteins.